WO2021180055A1 - 一种brd4抑制剂的用途 - Google Patents

一种brd4抑制剂的用途 Download PDF

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WO2021180055A1
WO2021180055A1 PCT/CN2021/079672 CN2021079672W WO2021180055A1 WO 2021180055 A1 WO2021180055 A1 WO 2021180055A1 CN 2021079672 W CN2021079672 W CN 2021079672W WO 2021180055 A1 WO2021180055 A1 WO 2021180055A1
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compound
tumor
pharmaceutically acceptable
acceptable salt
dose
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PCT/CN2021/079672
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English (en)
French (fr)
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姚雪坤
张宋安
张威
高天宁
申丽
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石药集团中奇制药技术（石家庄）有限公司
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Priority to CN202180017690.XA priority Critical patent/CN115190800A/zh
Publication of WO2021180055A1 publication Critical patent/WO2021180055A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A61K31/5513—1,4-Benzodiazepines, e.g. diazepam or clozapine
    • A61K31/5517—1,4-Benzodiazepines, e.g. diazepam or clozapine condensed with five-membered rings having nitrogen as a ring hetero atom, e.g. imidazobenzodiazepines, triazolam
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

  • the invention belongs to the field of medicine, and specifically relates to the application of a BRD4 inhibitor in the preparation of drugs for treating tumors, especially advanced solid tumors or refractory solid tumors, and a method for treating tumors with the inhibitor.
  • Tumor disease is one of the most common diseases in modern times and a common cause of unnatural death.
  • Tumors include benign tumors and malignant tumors.
  • the risk of malignant tumors is very high, and most of the malignant tumors are life-threatening.
  • the methods of treatment of malignant tumors include surgical resection and drug chemotherapy.
  • the existing treatment methods are difficult to produce good results for patients with various advanced tumors including triple-negative breast cancer (TNBC) and colorectal cancer (CRC). Curative effect.
  • TNBC triple-negative breast cancer
  • CRC colorectal cancer
  • TNBC triple negative breast cancer
  • TNBC triple negative breast cancer
  • AR androgen receptor
  • TNBC has a significant risk of metastasis within 5 years.
  • DFS 5-year disease-free survival
  • OS overall survival
  • triple-negative breast cancer is slightly more sensitive to chemotherapy, but the prognosis of conventional chemotherapy is poor and the 5-year survival rate is extremely low, especially for breast cancer.
  • Patients with susceptibility gene (BRCA) mutations have worse efficacy, and there is a very high unmet medical need in this field.
  • Colorectal cancer also known as colorectal cancer, is a heterogeneous malignant tumor that grows in the first six feet of the large intestine (colon) and the last 8-10 inches of the large intestine (rectum). It involves various molecular pathways and genetic/epigenetics. The change triggers the sequential transformation of normal mucosa into adenoma and then into cancer. It is a common malignant tumor in the gastrointestinal tract. The early symptoms are not obvious. As the tumor grows, it shows changes in bowel habits, blood in the stool, diarrhea, alternating diarrhea and constipation, and local abdominal pain; in the late stage, anemia, weight loss and other systemic symptoms symptom.
  • the age of onset of colorectal cancer in my country is mostly between 40 and 60 years old.
  • the average age of onset is 48.3 years old, which is 10 to 15 years earlier than Westerners. It is more common in young patients in my country than in Europe and the United States. Colorectal cancer under 30 years old Patients are not uncommon. Colorectal cancer is very dangerous. Because early detection is not timely, more than 80% of patients in my country are actually in the middle and late stages when they are discovered. Due to the loss of the best treatment opportunity, the 5-year survival rate of patients after diagnosis is very low; In our country, 1 person dies of colorectal cancer every 5 minutes.
  • Colorectal cancer is currently mainly treated with surgery, but the quality of life after surgery has also significantly decreased, including: decreased sexual function and excretory function, increased psychological disorders, and decreased social activities. Domestic surveys show that the survival rate of early colorectal cancer after surgery is more than 90-95%, while the late stage is only 5%. As the incidence of colorectal cancer is increasing year by year, and surgical treatment has reached a bottleneck, it is difficult to make a major breakthrough, especially for patients with limited physical conditions that cannot be treated by surgical resection and patients with recurrence and metastasis after surgery. It is particularly important.
  • Bromodomain and extra-terminal domain is a class of proteins with two bromodomains and one extra-terminal domain, which can recognize acetylated lysines in histones .
  • BET bromodomain and extra-terminal domain
  • BRD4 Bromodomain-containing protein 4
  • BRD4 protein combines with RNA polymerase II and forward transcription elongation factor to participate in the transcription process of oncogenes such as MYC, BCL2 and BCL6, and then regulate cell transcription.
  • BRD4 Marks the mitosis of mammals, regulates the cell cycle, and plays an important role in the occurrence and development of cancer, fibrosis and inflammation.
  • Highly expressed BRD4 has been detected in leukemia, lymphoma, breast cancer, gastrointestinal tumors and other blood system and solid tumor cancer cells and fibrotic cells.
  • tumor cell apoptosis that is, proliferation slowed, can be induced to achieve anti-tumor effects. Therefore, BRD4, as a promising anti-tumor target, has received extensive attention and development in recent years.
  • BRD4 inhibitors have become a hot field of drug research for solid tumors, leukemia and fibrosis.
  • BRD4 inhibitors currently have no drugs on the market at home and abroad, but many pharmaceutical companies have small molecule drugs in the clinical stage.
  • Merck's Birabresib MK-8628, OTX-015
  • GlaxoSmithKline GSK
  • GSK GlaxoSmithKline
  • AZ AstraZeneca
  • molibresib can be used to treat solid and blood cancers, including multiple myeloma, non-Hodgkin’s lymphoma, acute myeloid leukemia, small cell lung cancer, castration resistant prostate cancer, colorectal cancer, non-small cell lung cancer, and breast cancer Cancer (including metastatic breast cancer, estrogen receptor positive (ER+) breast cancer, and triple negative breast cancer (TNBC)).
  • Birabresib (MK8628) is undergoing clinical research including acute leukemia (AL) and other hematological malignancies (OHM), triple-negative breast cancer, non-small cell lung cancer, castration-resistant prostate cancer, and pancreatic ductal cancer.
  • Gastrointestinal toxicity is a prominent toxic reaction in the preclinical toxicological studies of BDR4 inhibitors, but the reaction varies greatly among different species. Among them, rats and dogs have poor tolerance, while human intestinal wall This is not sensitive. It is reported that MK-8628 has strong gastrointestinal toxicity when used in rats, but it is relatively well tolerated by humans in the clinical stage. The situation of AZ's BET inhibitor AZ5153 is similar. Common gastrointestinal adverse reactions include diarrhea, constipation, nausea, vomiting, dysgeusia, and mucositis. Hematological toxicity is also one of the main clinical adverse effects of BDR4 inhibitors.
  • BET inhibitors can inhibit miR17-92, thereby significantly up-regulating BIM protein, directly or indirectly activating BAX and BAK proteins, and then controlling internal mitochondrial cell apoptosis The process causes the death of normal hematopoietic cells.
  • Compound OTX015 and MK-8628 have been observed in clinical studies with blood system adverse reactions such as thrombocytopenia, anemia, and neutropenia.
  • Compound A is a small molecule inhibitor of BRD4, which can significantly inhibit the activity of BRD4 protein and is expected to be used in the treatment of tumors. Its structure is shown in the following formula (I):
  • WO2019056950A1 discloses the preparation method and activity of compound A, and WO2020192637A1 further discloses the crystal form and use of compound A.
  • Compound A currently does not have sufficient pharmacodynamic and safety evaluation data, so the important risks and exact efficacy of its clinical application are still unclear.
  • the present invention explores the effectiveness and safety of compound A in the treatment of tumors, in order to provide a reference for clinical medication.
  • the purpose of the present invention is to provide the use of compound A or a pharmaceutically acceptable salt thereof in the preparation of drugs for the treatment of tumors, wherein the tumors are preferably advanced solid tumors or refractory solids Tumor, the structure of the compound A is shown in the following formula (I):
  • the solid tumor in the advanced solid tumor or refractory solid tumor is gastrointestinal tumor, colorectal cancer, breast cancer, or prostate cancer.
  • the solid tumors in the advanced solid tumors or refractory solid tumors are colorectal cancer with high infiltration of tumor-associated macrophages, triple-negative breast cancer, and androgen-independent prostate cancer.
  • the aforementioned subject suffering from the tumor is a human.
  • the drug contains a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof.
  • the therapeutically effective amount is preferably about 0.001 to about 1000 mg.
  • the administration mode of Compound A or a pharmaceutically acceptable salt thereof is not particularly limited, and the representative administration mode may be oral administration, injection administration, topical administration or in vitro administration.
  • the medicine uses conventional auxiliary materials and techniques to prepare clinically accepted preparations, such as oral preparations, injection preparations, topical administration preparations, topical preparations, and the like.
  • the drug is in a single-dose dosage form or a multiple-dose dosage form.
  • the dosage form contains about 0.001 mg to about 1000 mg of compound A or a pharmaceutically acceptable salt thereof (calculated as compound A), preferably about 1 mg to about 500 mg, or about 5 mg to about 400 mg, or about 10 mg to about 300 mg, or About 10 mg to about 250 mg, or about 10 mg to about 200 mg, or about 20 mg to about 200 mg, more preferably about 10 mg to about 160 mg, or about 20 mg to about 160 mg, or about 80 mg to about 160 mg; for example, about 10 mg, about 20 mg, about 40 mg, about 60 mg, about 80 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg, more preferably about 10 mg, about 20 mg, about 40 mg, about 80 mg, about 120 mg, or about 160 mg, still more preferably about 20 mg, about 40 mg, about 80 mg, about 120 mg, or about 160 mg, or further more preferably about 10 mg or about 40 mg.
  • Compound A or a pharmaceutically acceptable salt thereof may be used in combination with one or more of other targeted drugs or chemotherapeutic drugs.
  • the other targeted drugs or chemotherapeutics refer to targeted drugs or chemotherapeutics that are clinically used to treat tumor-related diseases.
  • the object of the present invention is to provide a method for treating tumors, the method comprising administering to a subject or patient a therapeutically effective amount of Compound A or a pharmaceutically acceptable salt thereof, wherein the tumor is an advanced entity Tumor or refractory solid tumor.
  • the solid tumor in the advanced solid tumor or refractory solid tumor is gastrointestinal tumor, breast cancer, colorectal cancer or prostate cancer.
  • the solid tumors in the advanced solid tumors or refractory solid tumors are colorectal cancer with high infiltration of tumor-associated macrophages, triple-negative breast cancer, and androgen-independent prostate cancer.
  • the subject or patient is a human.
  • a suitable dosage range of Compound A or a pharmaceutically acceptable salt thereof is from about 0.001 mg/kg to about 1000 mg/kg per day; preferably, from about 0.01 mg/kg to about 100 mg/kg.
  • the daily dose of Compound A or its pharmaceutically acceptable salt is about 0.001 mg to about 1000 mg. It is administered in a single dose or in divided doses.
  • the above-mentioned administration is oral administration, injection administration, topical administration or in vitro administration; or compound A or a pharmaceutically acceptable salt thereof is prepared into a clinically accepted preparation, and the preparation includes oral preparation, injection preparation , Local administration preparations, topical preparations.
  • Compound A or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 0.001 mg to about 1000 mg, preferably about 1 mg to about 500 mg, or about 5 mg to about 400 mg, or about 10 mg to about 300 mg, or about 10 mg to about 250 mg, or about 10 mg to about 200 mg, or about 20 mg to about 200 mg; more preferably about 10 mg to about 160 mg, or about 20 mg to about 160 mg, or about 80 mg to about 160 mg; for example, about 10mg, about 20mg, about 40mg, about 60mg, about 80mg, about 100mg, about 120mg, about 140mg, about 160mg, more preferably about 10mg, about 20mg, about 40mg, about 80mg, about 120mg or about 160mg, and more preferably about 20 mg, about 40 mg, about 80 mg, about 120 mg, or about 160 mg, administered in a single dose or divided doses.
  • the dosage regimen of Compound A or its pharmaceutically acceptable salt is 28 days per cycle, once a day for 21 days, with 7 days off; preferably, compound A or its pharmaceutically acceptable salt is administered every time
  • the pharmaceutically acceptable salt (calculated as compound A) is about 20 mg, about 40 mg, about 80 mg, about 120 mg, and about 160 mg.
  • the compound A or a pharmaceutically acceptable salt thereof is used in combination with one or more of other targeted drugs or chemotherapeutic drugs.
  • the purpose of the present invention is to provide compound A or a pharmaceutically acceptable salt thereof for the treatment of tumors, preferably advanced solid tumors or refractory solid tumors.
  • the solid tumor in the advanced solid tumor or refractory solid tumor is gastrointestinal tumor, colorectal cancer, breast cancer or prostate cancer.
  • the solid tumors in the advanced solid tumors or refractory solid tumors are colorectal cancer with high infiltration of tumor-associated macrophages, triple-negative breast cancer, and androgen-independent prostate cancer.
  • the subject suffering from the tumor is a human.
  • the compound A or a pharmaceutically acceptable salt thereof is prepared into a clinically accepted preparation and then administered.
  • the preparation includes an oral preparation, an injection preparation, a topical administration preparation, and an external preparation.
  • Compound A or a pharmaceutically acceptable salt thereof is administered in a daily dose of about 0.001 mg to about 1000 mg, preferably about 1 mg to about 500 mg, or about 5 mg to about 400 mg, or about 10 mg to about 300 mg, or about 10 mg to about 250 mg, or about 10 mg to about 200 mg, or about 20 mg to about 200 mg; more preferably about 10 mg to about 160 mg, or about 20 mg to about 160 mg, or about 80 mg to about 160 mg; for example, about 10mg, about 20mg, about 40mg, about 60mg, about 80mg, about 100mg, about 120mg, about 140mg, about 160mg, more preferably about 10mg, about 20mg, about 40mg, about 80mg, about 120mg or about 160mg, and more preferably about 20 mg, about 40 mg, about 80 mg, about 120 mg, or about 160 mg, administered in a single dose or divided doses.
  • the dosage regimen of Compound A or its pharmaceutically acceptable salt is 28 days per cycle, once a day for 21 days, with 7 days off; preferably, compound A or its pharmaceutically acceptable salt is administered every time
  • the pharmaceutically acceptable salt (calculated as compound A) is about 20 mg, about 40 mg, about 80 mg, about 120 mg, and about 160 mg.
  • the compound A or a pharmaceutically acceptable salt thereof can be used in combination with one or more of other targeted drugs or chemotherapeutics.
  • the dosage of Compound A or its pharmaceutically acceptable salt of the present invention is all calculated as Compound A.
  • Advanced solid tumor refers to cancer that has spread outside the origin or organ through local invasion or metastasis.
  • advanced solid tumor includes locally advanced and metastatic solid tumors.
  • Refractory solid tumor refers to a solid tumor whose condition is still progressing even if anti-tumor therapy is used on the patient.
  • the anti-tumor therapy includes radiotherapy, chemotherapy drug therapy, targeted drug therapy, immune checkpoint drug therapy and the like.
  • the refractory solid tumor refers to a solid tumor for which standard treatment regimens are ineffective.
  • Figure 1 is a flow chart of the Phase I clinical program.
  • the compound dilution was completed by the Echo non-contact nano-upgraded sonic pipetting system, which was diluted by 3 times to 10 concentrations: 20000, 6666.67, 2222.22, 740.74, 246.91, 82.305, 27.435, 9.145, 3.048, 1.016nM.
  • test buffer solution 1 ⁇ assay buffer (test buffer solution) to configure the "solution A” (protein solution), “solution B” (peptide solution), and “solution C” (test reagent solution) used in the experiment, and make each The components form a 3 ⁇ solution in the experimental reaction system, and the amount of solutions A, B, and C must be sufficient for this round of experiments.
  • the experiment board is the board containing the gradient concentration of the compound and the corresponding DMSO solution prepared by the Echo non-contact nano-upgraded sonic pipetting system before the experiment:
  • Compound A has a significant inhibitory effect on BRD4-BD1 and BRD4-BD2.
  • Example 2 In vivo pharmacodynamic study of compound A in human breast cancer MDA-MB-231_luc cell subcutaneous xenograft tumor model
  • Week age and weight 6-8 weeks old, weighing 18-22 grams
  • Human breast cancer MDA-MB-231_luc cells are cultured in a monolayer in vitro, and the culture conditions are RPMI-1640 medium (supplier: Gibco; article number: 22400-089; production lot number: 4868546) with 10% fetal bovine serum, 100U/ml Penicillin and 100 ⁇ g/ml streptomycin were cultured at 37°C with 5% CO 2.
  • RPMI-1640 medium supplier: Gibco; article number: 22400-089; production lot number: 4868546
  • 10% fetal bovine serum 100U/ml Penicillin and 100 ⁇ g/ml streptomycin were cultured at 37°C with 5% CO 2.
  • pancreatin-EDTA for routine digestion and passage twice a week.
  • the cells are in the exponential growth phase, the cells are collected, counted, and seeded.
  • the experimental index is to investigate whether the tumor growth is inhibited, delayed or cured.
  • the tumor diameter was measured with vernier calipers twice a week.
  • the anti-tumor efficacy of the compound was evaluated by TGI (%) or the relative tumor growth rate T/C (%).
  • RTV relative tumor volume
  • Example 3 In vivo pharmacodynamic study of compound A in human prostate cancer PC-3 cell subcutaneous xenograft tumor model
  • the preparation method of the test substance is the same as Table 2, and the animal grouping and dosing schedule are the same as Table 3.
  • Week age and weight 6-8 weeks old, weighing 18-22 grams
  • Human prostate cancer PC-3 cells were cultured in a monolayer in vitro with F-12K medium (supplier: Gibco; article number: 21127-022; production lot number: 1868870) with 10% fetal bovine serum and 100U/mL penicillin Incubate with 100 ⁇ g/mL streptomycin at 37°C with 5% CO 2 . Use pancreatin-EDTA for routine digestion and passage twice a week. When the cells are in the exponential growth phase, the cells are collected, counted, and seeded.
  • Example 4 In vivo anti-tumor efficacy study of Compound A in MC38 mouse colon cancer cell animal transplantation tumor model
  • the preparation method of the test substance is the same as Table 2, and the animal grouping and dosing schedule are shown in Table 4 below.
  • Week age and weight 6-7 weeks old, weighing 16-20 grams
  • Mouse colon cancer MC38 cells (Heyuan Biotechnology Co., Ltd.) were cultured in a monolayer in vitro with 10% fetal bovine serum in DMEM medium (Gibco, article number: 12100), 37°C and 5% CO 2 in an incubator nourish. Use 0.25% pancreatin-EDTA for routine digestion and passage. When the cells are in the exponential growth phase and the saturation is 80% to 90%, the cells are collected, counted, and inoculated.
  • 0.1 mL of 2 ⁇ 10 5 MC38 cells were subcutaneously inoculated on the right back of each mouse. When the average tumor volume reached about 70 mm 3 , randomized administration was performed according to the tumor volume.
  • the single dose of compound A in group A is 100 mg/kg, the single dose volume is 10 mL/kg, and the dose concentration is 10 mg/mL (maximum gavage concentration). The dose is administered twice within 24 hours (8 hours apart). The total dosage is 200 mg/kg.
  • the vehicle control group was given an equal volume of vehicle.
  • the animals in the compound A administration group had no symptoms of severe poisoning, and there were no abnormalities in food intake, blood index, coagulation index, and gross anatomical observation. 200mg/kg of compound A may have a reversible toxic effect on the digestive system of SD rats, which can cause animal salivation, watery stools and slightly slower weight gain in females. The serum UREA level of male animals in the compound A administration group decreased. Therefore, under the conditions of this experiment, the maximum tolerated dose (MTD) of compound A administered by oral gavage to SD rats was 200 mg/kg.
  • MTD maximum tolerated dose
  • the groups are: vehicle control group, compound A low-dose group, compound A medium-dose group, and compound A high-dose group.
  • the single doses of the compound low, medium and high dose groups were 6mg/kg, 18mg/kg and 48mg/kg, and they were given twice within 24 hours (8 hours apart), and the total doses were 12mg/kg and 36mg respectively. /kg and 96mg/kg.
  • the vehicle control group was given an equal volume of vehicle.
  • the animals in each administration group of Compound A showed no symptoms of severe poisoning, and only transient watery stools appeared after the second administration on the day of administration.
  • the animals in the high-dose group had a decrease in food intake 4 to 5 days after administration. the trend of.
  • the blood indexes WBC and #NEUT of Beagle dogs in each dose group of compound A showed an increasing trend after administration. Therefore, under the experimental conditions, the non-toxic reaction dose (NOAEL) of compound A to Beagle dogs in a single administration is 36 mg/kg; MTD is 96 mg/kg.
  • NOAEL non-toxic reaction dose
  • Example 7 SD rats repeated administration for 4 weeks and recovery for 4 weeks accompanied by TK toxicity test study
  • SD rats were divided into 4 groups, each with 46 rats/group (main test 30 rats/group, TK satellite group 16 rats/group), half male and female.
  • the groups were vehicle control group, compound A low-dose group, compound A medium-dose group, and compound A high-dose group.
  • SD rats in the low, medium, and high dose groups of compound A were given 2, 6 and 20 mg/kg of compound A by oral gavage twice a day (8 hours apart) (total doses of 4, 12, and 40 mg/kg, respectively. day) for 4 consecutive weeks, stop the drug and resume for 4 weeks.
  • the vehicle control group was given an equal volume of vehicle.
  • TK results showed that after D1 administration, there was a significant gender difference in the AUC0-24h dose group of compound A.
  • the system exposure (AUC0-24h and Cmax) of the other dose groups had no significant gender difference;
  • D1 After administration with D28, the systemic exposure (AUC0-24h and Cmax) of compound A in female animals increased in a dose-proportional manner;
  • the increase in AUC0-24h of compound A in male animals was higher than the increase in dose, and Cmax increased in a dose-proportional manner; It was administered twice a day, and after continuous administration for 4 weeks, there was no accumulation of compound A exposure in the plasma of female and male SD rats.
  • the MTD is 40 mg/kg/day.
  • the AUC0-24h of male and female animals on the 28th day were 23115 and 18017h*nM, respectively.
  • NOAEL is 4mg/kg/day.
  • Example 8 Beagle dog repeated administration for 28 days, recovery for 28 days with TK toxicity test study
  • Beagle dogs were randomly divided into 4 groups, 10 per group, half male and half female.
  • the groups were vehicle control group, compound A low-dose group, compound A medium-dose group, and compound A high-dose group.
  • Compound A low, medium, and high dose groups were given 1, 3, and 6 mg/kg of Compound A by oral gavage twice a day (8 hours apart) (total daily doses were 2 mg/kg, 6 mg/kg, and 12 mg/kg, respectively). kg) for 4 consecutive weeks, and 4 weeks of recovery after stopping the drug.
  • High-dose and/or medium-dose Compound A affects Beagle dog hematology indexes (reduction of RETIC, EOS, BASO), coagulation indexes (prolonged APTT, increase of FIB) and blood biochemical indexes (TP, ALB, CREA, UREA decrease, ALT, AST, CHOL, TG, GLU increased), but the effect is reversible. Histopathological examination revealed that 2 mg/kg of compound A had a reversible toxic effect on the immune system (thymus) of Beagle dogs.
  • Compound A at 6-12 mg/kg has reversible toxic effects on the digestive system (pancreas, liver, digestive tract), immune system (thymus/spleen, intestinal-related lymph nodes), blood system (sternal bone marrow) and skin of Beagle dogs.
  • the toxic effects of the reproductive system (testis, epididymis) of Beagle dogs were not fully recovered after the drug was stopped, and extramedullary hematopoiesis could be seen in the spleen in the convalescent animals.
  • Compound A at 12 mg/kg has a reversible toxic effect on the respiratory system (trachea, lung) of Beagle dogs.
  • TK results showed that after D1 and D28 administration, there was no significant gender difference in the compound A system exposure (AUC0-24h and Cmax) of each dose group; after repeated administration, the compound A exposure (AUC0-24h and Cmax) of each dose group were all No significant accumulation; after the administration of D1 and D28, the increase ratio of AUC0-24h of compound A was higher than the increase in dose, and Cmax both increased in a dose-proportional manner.
  • the lethal dose is 12 mg/kg/day
  • the HNSTD the highest non-seriously toxic dose
  • the 28th day (AUC0-24h) of male and female animals were 9443.7 and 9149.9h*nM, respectively.
  • the lowest dose (LOAEL) at which adverse effects were observed was 2 mg/kg/day.
  • Safety the occurrence and frequency of AE, SAE, and DLT; determine the maximum tolerated dose (MTD) (if any) of compound A, phase II clinical recommended dose (RP2D) and dosing schedule.
  • MTD maximum tolerated dose
  • Pharmacokinetic indicators including but not limited to AUC 0-last , AUC 0- ⁇ , C max , T max , t 1/2 and CL/F, etc.
  • Curative effect indicators total remission rate (ORR), progression-free survival (PFS), disease control rate (DCR), duration of remission (DOR), etc.
  • the first stage (Stage I):
  • the initial dose of compound A was set at 20 mg/day, and 5 dose groups were initially proposed: 20, 40, 80, 120, and 160 mg.
  • the preset maximum escalating dose is 160 mg/day. This study will follow the "i3+3" dose escalation schedule.
  • the first cycle of each dose level (31 days), after the subject's single dose on the first day (day 1), if there is no DLT or serious adverse events on the 3rd day of observation, the subject will enter the continuous dosing phase on the 4th day .
  • the specific dosing frequency will be adjusted according to the patient's tolerance, safety and drug PK parameters).
  • the target dose group dose expansion study and the exploratory study of different dosing schemes can be carried out.
  • Sponsors and investigators will continue to conduct safety evaluations, and based on the available data of previous dose levels, determine the dose level and dosing schedule during dose escalation and dose expansion.
  • a cohort expansion study will be conducted in different indications to observe the safety, tolerability, pharmacokinetic characteristics and resistance of the study drug. Tumor activity.
  • each expansion group is expected to enroll 20 to 40 subjects.
  • the sample size and tumor types for the final cohort expansion will be determined after full discussion with the investigator after comprehensive consideration of the first phase and preclinical research data. Moreover, sponsors and investigators may consider ending the enrollment of a cohort early based on the principle of the maximum benefit/risk ratio of the patient.
  • Enrolled subjects must have advanced or metastatic tumors diagnosed by histology or cytology, and have no standard treatment plan, or those who are ineffective or intolerant to the standard treatment plan, or have no conditions to receive standard treatment and meet Requirements for tumor types at the following corresponding stages:
  • the estimated survival time is more than 3 months.
  • glucocorticoids prednisone> 10 mg/day or equivalent doses of similar drugs
  • other immunosuppressive agents within 14 days before the first use of the study drug; except for the following cases: use of local or eye , Intra-articular, intranasal and inhaled glucocorticoid therapy; short-term use of glucocorticoids for preventive treatment (for example, prevention of contrast agent allergy).
  • hepatitis B hepatitis B virus titer>1000 copies/ml or 200IU/ml
  • hepatitis C antibody is positive.
  • Severe heart rhythm or conduction abnormalities such as ventricular arrhythmia that requires clinical intervention, degree II to III atrioventricular block, etc., or other arrhythmias that require antiarrhythmic drugs (excluding anticoagulant drugs) ,
  • the coagulation function related to taking anticoagulant drugs must meet the selection criteria);
  • Baseline echocardiography (ECHO) or multi-gate acquisition (MUGA) technology shows left ventricular ejection fraction (LVEF) ⁇ 50%;
  • the expected maximum tolerated dose (MTD) of compound A is 120mg/d ⁇ 160mg/d, and the expected therapeutic dose is 80mg/d.
  • MTD maximum tolerated dose
  • compound A administered at 80 mg/d, 120 mg/d, and 160 mg/d will have an effective therapeutic effect on patients with advanced solid tumors, especially gastrointestinal tumors, triple-negative breast cancer, colorectal cancer, and prostate cancer. .
  • Compound A will be able to safely and effectively treat advanced solid tumors, especially gastrointestinal tumors, triple negative breast cancer, colorectal cancer, and prostate cancer.
  • the patient was given Compound A orally for the first time on 2020-5-13, with a dose of 20 mg. No serious abnormalities were observed for two days, and the administration was continued on 2020-5-15, the dose was 20 mg/day, and the last dose was 2020-7-10, for a total of 2 cycles (28 days per cycle).
  • an enhanced CT examination was performed, and the comprehensive assessment was SD (stable disease).
  • grade 1 hypoalbuminemia, grade 1 anemia, and grade 1 white blood cell count decreased. Hypoproteinemia may not be related to the study drug, and the latter two may be related to the study drug.

Abstract

一种BDR4抑制剂在制备***、尤其是治疗晚期实体瘤或难治性实体瘤的药物中的应用，该药物治疗晚期实体瘤或难治性实体瘤的治疗方案和安全有效剂量。该BDR4抑制剂能有效治疗晚期实体瘤或难治性实体瘤，对晚期或难治性的乳腺癌、结直肠癌、***癌、胃肠肿瘤等有确切疗效，并且临床安全性较好。

Description

一种BRD4抑制剂的用途
本申请要求2020年03月09日向中国国家知识产权局提交的申请号为202010156960.2，发明名称为“一种BDR4抑制剂的用途”的在先发明专利申请的优先权。该在先申请的全文通过引用的方式纳入本申请中。
技术领域
本发明属于医药领域，具体涉及一种BRD4抑制剂在制备***、尤其是晚期实体瘤或难治性实体瘤的药物中的应用，以及该抑制剂***的方法。
背景技术
肿瘤疾病是现代最常见的疾病之一，也是非自然死亡的常见原因。肿瘤包括良性肿瘤和恶性肿瘤两种类型，恶性肿瘤的危险性非常高，大部分的恶性肿瘤都会危及生命。目前，治疗恶性肿瘤的方法包括手术切除以及药物化疗，现有的治疗手段，对包括三阴乳腺癌(TNBC)、结直肠癌(CRC)在内的多种中晚期肿瘤患者难以产生较好的疗效。
根据最新的数据，2018年全球新发肿瘤病例约1800万，其中乳腺癌占11.6％，位居全球第二；因癌症死亡人数接近960多万，乳腺癌占比6.6％，位居全球第六。在国内，乳腺癌的发病率为45.29/100000，发病人数位居全国第五。同时乳腺癌已成为国内女性第一高发的恶性肿瘤类型，我国女性每年约有1.6万人因乳腺癌死亡，乳腺癌已成为我国女性第5位恶性肿瘤死亡原因。三阴性乳腺癌(TNBC)是指***受体(ER)、孕激素受体(PR)和人表皮生长因子受体(HER-2)均为阴性的乳腺癌，在所有乳腺癌病例中约占15％。三阴乳腺癌在亚洲人和非裔美国人等特定种族群体中发病率较高。TNBC患者具有年轻化、家族性、侵袭性、复发性和转移性等特点。研究表明TNBC中有87％的患者呈雄激素受体(androgen receptor,AR)阴性表达，导致其易于复发及发生远处转移，与其它类型的乳腺癌相比，TNBC在5年内转移的风险明显更大，转移扩散主要发生在内脏器官，尤其是肺及脑部转移。5年无病生存期(DFS)及总生存期(OS)也均显著低于非三阴乳腺癌。现有乳腺癌靶向药物对三阴乳腺癌无效，与其他类型乳腺癌相比，三阴乳腺癌对化疗敏感性略高，但常规化疗预后差、5年生存率极低，特别是乳腺癌易感基因(BRCA)突变的患者疗效更差，该领域存在非常高的未满足的医疗需求。
结直肠癌又称大肠癌，是一种生长在大肠的前六英尺(结肠)及大肠的最后8～10英寸(直肠)的异质性恶性肿瘤，涉及各种分子途径和遗传/表观遗传改变，触发正常粘膜顺序 转化为腺瘤，然后转变为癌。它是胃肠道中常见的恶性肿瘤，早期症状不明显，随着肿瘤的增大而表现排便***均发病年龄为48.3岁，比西方人足足早了10～15年，在我国青年患者比欧美更为多见，30岁以下的大肠癌患者并不少见。结直肠癌危害性非常大，由于早期发现不及时，在我国实际上超过80％患者发现时已是中晚期，由于失去了最佳的治疗时机，导致患者确诊后5年生存率很低；在我国每5分钟就有1人死于结直肠癌。结直肠癌目前主要采用外科手术治疗，但是采取外科术后生活质量也明显下降，包括：性功能、***功能下降，心理障碍增加，社交活动减少等。国内调查显示，早期结直肠癌术后存活率达90～95％以上，而晚期则只有5％。随着结直肠癌发病率逐年上升，且外科手术治疗已经达到一个瓶颈，再难有大的突破，特别是对于身体条件受限无法通过手术切除治疗的患者以及术后复发转移的患者，药物治疗显得尤为重要。
Bromodomain and extra-terminal domain，即溴结构域及超末端结构家族(BET)，是一类具有两个溴结构域及一个超末端结构域的蛋白，可以识别组蛋白中的乙酰化的赖氨酸。在人体中有4种BET家族成员:BRD2，BRD3，BRD4及BRDT。其中，BRD4(Bromodomain-containing protein 4)在人体中表达极为广泛，BRD4蛋白通过与RNA聚合酶II，正向转录延长因子结合共同参与MYC、BCL2和BCL6等癌基因的转录过程，继而调节细胞转录，标记哺乳动物的有丝***，调控细胞周期，在癌症的发生、发展，纤维化及炎症过程中发挥着重要的作用。在白血病、淋巴瘤、乳腺癌、胃肠道肿瘤等血液***和实体肿瘤的癌细胞及纤维化细胞中，均检测到高表达的BRD4。通过靶向抑制BRD4，可以诱发肿瘤细胞凋亡即增殖减缓，从而达到抗肿瘤作用。因此，BRD4作为一种极具潜力的抗肿瘤靶点，在近些年得到了广泛关注和开发。BRD4抑制剂已成为实体瘤、白血病及纤维化等疾病药物研究的热门领域。
BRD4抑制剂目前国内外还没有上市药物，但已有多个制药公司的小分子药物处于临床阶段。例如Merck公司的Birabresib(MK-8628，OTX-015)、GlaxoSmithKline(GSK)正在开发的molibresib(GSK-525762A，GSK-525762，I-BET-762，GSK'762)、阿斯利康(AZ)在研的AZ5153等。据报道，molibresib可用于治疗实体和血液癌症，包括多发性骨髓瘤、非霍奇金淋巴瘤、急性髓性白血病、小细胞肺癌、去势抵抗***癌、结直肠癌、非小细胞肺癌、乳腺癌(包括转移性乳腺癌、***受体阳性(ER+)乳腺癌及三阴性乳腺癌(TNBC))。Birabresib(MK8628)正在进行包括急性白血病(AL)和其他血液***恶性 肿瘤(OHM)、三阴性乳腺癌、非小细胞肺癌、去势抵抗性***癌和胰腺导管癌的临床研究。
胃肠道毒性是BDR4抑制剂临床前毒理研究中较为突出的毒性反应，但该反应在不同种属之间差异大，其中大鼠和犬耐受性都较差，而人的肠壁对此并不敏感。据报道，MK-8628用于大鼠有很强的胃肠道毒性，但临床阶段人的耐受性相对较好。AZ在研的BET抑制剂AZ5153情况类似。常见的胃肠道不良反应有腹泻、便秘、恶心、呕吐、味觉障碍、粘膜炎等。血液毒性也是BDR4抑制剂临床主要不良反应之一，有研究发现BET抑制剂会抑制miR17-92，从而使得BIM蛋白显著上调，直接或间接的活化BAX及BAK蛋白，进而通过控制内在线粒体细胞凋亡过程，使得正常的造血细胞死亡。化合物OTX015、MK-8628在临床研究中都观察到了血小板减少症、贫血症、中性粒细胞减少症等血液***不良反应。
化合物A是一种BRD4小分子抑制剂，能显著抑制BRD4蛋白的活性，有望用于***，其结构如下式(Ⅰ)所示：
Figure PCTCN2021079672-appb-000001
WO2019056950A1公开了化合物A的制备方法和活性，WO2020192637A1进一步公开了化合物A的晶型和用途。化合物A目前尚无充分的药效和安全性评价数据，因此尚不清楚其用于临床的重要风险和确切疗效。本发明探讨化合物A***的有效性和安全性，以期为临床用药提供参考。
发明内容
基于上述现有技术缺陷，第一方面，本发明的目的在于提供化合物A或其药学上可接受的盐在制备***的药物中的应用，其中所述肿瘤优选晚期实体瘤或难治性实体瘤，所述化合物A的结构如下式(Ⅰ)所示：
Figure PCTCN2021079672-appb-000002
在一些实施方式中，所述晚期实体瘤或难治性实体瘤中的实体瘤为胃肠肿瘤、结直肠癌、乳腺癌或***癌等。
在一些实施方式中，所述晚期实体瘤或难治性实体瘤中的实体瘤为肿瘤相关巨噬细胞高度浸润的结直肠癌、三阴乳腺癌、雄激素非依赖型***癌。
在一些实施方式中，上述患有所述肿瘤的对象为人。
在一些实施方式中，所述药物含有治疗有效量的化合物A或其药学上可接受的盐。所述治疗有效量优选约0.001-约1000mg。
在一些实施方式中，化合物A或其药学上可接受的盐的给药方式没有特别限定，代表性的给药方式可以是口服给予、注射给予、局部给予或体外给予。相应的，所述药物使用常规辅料和工艺制成临床接受的制剂，例如口服制剂、注射制剂、局部给药制剂、外用制剂等。所述药物为单剂量剂型或多剂量剂型。所述剂型含有约0.001mg-约1000mg的化合物A或其药学上可接受的盐(以化合物A计)，优选约1mg-约500mg，或约5mg-约400mg，或约10mg-约300mg，或约10mg-约250mg，或约10mg-约200mg，或约20mg-约200mg，更优选约10mg-约160mg，或约20mg-约160mg，或者约80mg-约160mg；例如约10mg、约20mg、约40mg、约60mg、约80mg、约100mg、约120mg、约140mg、约160mg，更优选约10mg、约20mg、约40mg、约80mg、约120mg或约160mg，进一步更优选约20mg、约40mg、约80mg、约120mg或约160mg，或者进一步更优选约10mg或约40mg。
在一些实施方式中，化合物A或其药学上可接受的盐可与其他靶向药物或化疗药物中的一种或多种联合使用。所述的其他靶向药物或化疗药物是指临床用于***相关疾病的靶向药物或化疗药物。
第二方面，本发明的目的在于提供一种***的方法，所述方法包括给予受试者或患者治疗有效量的化合物A或其药学上可接受的盐，其中，所述肿瘤为晚期实体瘤或难治性实体瘤。
在一些实施方式中，所述晚期实体瘤或难治性实体瘤中的实体瘤为胃肠肿瘤、乳腺癌、结直肠癌或***癌。
在一些实施方式中，所述晚期实体瘤或难治性实体瘤中的实体瘤为肿瘤相关巨噬细胞高度浸润的结直肠癌、三阴乳腺癌、雄激素非依赖型***癌。
在一些实施方式中，所述受试者或患者为人。
化合物A或其药学上可接受的盐合适的剂量范围为每天从约0.001mg/kg至约1000mg/kg；优选的，从约0.01mg/kg至约100mg/kg。优选的，化合物A或其药学上可接受的盐每天给药剂量为约0.001mg-约1000mg。以单剂量或分剂量施用。
在一些实施方式中，上述给予是口服给予、注射给予、局部给予或体外给予；或者化合物A或其药学上可接受的盐制成临床接受的制剂后给予，所述制剂包括口服制剂、注射制剂、局部给药制剂、外用制剂。
在一些实施方式中，化合物A或其药学上可接受的盐(以化合物A计)每天给药剂量为约0.001mg-约1000mg，优选约1mg-约500mg，或约5mg-约400mg，或约10mg-约300mg，或约10mg-约250mg，或约10mg-约200mg，或约20mg-约200mg；更优选约10mg-约160mg，或约20mg-约160mg，或者约80mg-约160mg；例如约10mg、约20mg、约40mg、约60mg、约80mg、约100mg、约120mg、约140mg、约160mg，更优选约10mg、约20mg、约40mg、约80mg、约120mg或约160mg，进一步更优选约20mg、约40mg、约80mg、约120mg或约160mg，以单剂量或分剂量施用。
在一些实施方式中，化合物A或其药学上可接受的盐的给药方案为每个周期28天，每天给药一次，给药21天，停药7天；优选每次给予化合物A或其药学上可接受的盐(以化合物A计)约20mg、约40mg、约80mg、约120mg、约160mg。
在一些实施方式中，所述化合物A或其药学上可接受的盐与其他靶向药物或化疗药物中的一种或多种联合使用。
第三方面，本发明的目的在于提供化合物A或其药学上可接受的盐，用于***，所述肿瘤优选晚期实体瘤或难治性实体瘤。
在一些实施方式中，所述晚期实体瘤或难治性实体瘤中的实体瘤为胃肠肿瘤、结直肠癌、乳腺癌或***癌。
在一些实施方式中，所述晚期实体瘤或难治性实体瘤中的实体瘤为肿瘤相关巨噬细胞高度浸润的结直肠癌、三阴乳腺癌、雄激素非依赖型***癌。
在一些实施方式中，患有所述肿瘤的对象为人。
在一些实施方式中，所述化合物A或其药学上可接受的盐制成临床接受的制剂后给予，所述制剂包括口服制剂、注射制剂、局部给药制剂、外用制剂。
在一些实施方式中，化合物A或其药学上可接受的盐(以化合物A计)每天给药剂量为约0.001mg-约1000mg，优选约1mg-约500mg，或约5mg-约400mg，或约10mg-约300mg，或约10mg-约250mg，或约10mg-约200mg，或约20mg-约200mg；更优选约10mg-约160mg，或约20mg-约160mg，或者约80mg-约160mg；例如约10mg、约20mg、约40mg、约60mg、约80mg、约100mg、约120mg、约140mg、约160mg，更优选约10mg、约20mg、约40mg、约80mg、约120mg或约160mg，进一步更优选约20mg、约40mg、约80mg、约120mg或约160mg，以单剂量或分剂量施用。
在一些实施方式中，化合物A或其药学上可接受的盐的给药方案为每个周期28天，每天给药一次，给药21天，停药7天；优选每次给予化合物A或其药学上可接受的盐(以化合物A计)约20mg、约40mg、约80mg、约120mg、约160mg。
在一些实施方式中，所述化合物A或其药学上可接受的盐可与其他靶向药物或化疗药物中的一种或多种联合使用。
本发明所述的化合物A或其药学上可接受的盐的剂量，均以化合物A计。
为评价化合物A或其药学上可接受的盐***的疗效和安全性，本申请的发明人开展BDR4体外活性抑制试验、体内抑瘤试验、临床Ⅰ期研究，结果显示化合物A作用机理明确，能显著抑制BRD4蛋白的活性，在结肠癌、***癌、三阴乳腺癌小鼠体内药效模型中抑制肿瘤生长效果明显。临床拟用于治疗晚期实体瘤，包括但不限于胃肠肿瘤、结直肠癌、三阴乳腺癌、***癌等。可见，化合物A能有效治疗晚期实体瘤，尤其是胃肠肿瘤、三阴乳腺癌、结直肠癌、***癌，临床安全性良好。
术语解释：
“晚期实体瘤”是指已经通过局部侵入或转移在起源的部位或器官外部扩散的癌症。术语“晚期实体瘤”包括局部晚期和转移性实体瘤。
“难治性实体瘤”是指即使对患者使用抗肿瘤治疗，病情仍有进展的实体瘤。所述抗肿瘤治疗包括放射治疗、化疗药物治疗、靶向药物治疗、免疫检查点药物治疗等。在一些实施例中，所述难治性实体瘤是指标准治疗方案无效的实体瘤。
附图说明
图1为临床Ⅰ期方案流程图。
具体实施方式
以下列举的实施例是为了更好地对本发明的内容进行说明，但并不是本发明的内容仅限于所举实施例。本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整，仍属于本发明的保护范围。
一、化合物A对BRD4体外活性抑制试验
实施例1：BRD4生化活性检测
1.实验准备：
1)实验使用BPS公司的BRD4-BD1和BRD4-BD2蛋白；ANASPEC公司的多肽；PerkinElmer公司的检测试剂；
2)实验应用TR-FRET的实验原理对化合物进行筛选。
3)相关对照化合物
2.实验步骤：
1)准备化合物板：
实验中的化合物板的准备通过Echo非接触式纳升级声波移液***实现：
化合物稀释由Echo非接触式纳升级声波移液***完成，3倍递减稀释成10个浓度:20000、6666.67、2222.22、740.74、246.91、82.305、27.435、9.145、3.048、1.016nM。
2)反应试剂的准备：
相关的反应试剂应在实验当天准备：
a)配制1×assay buffer(测试缓冲溶液)；
b)配制3×的实验用组分溶液：
1.将试剂取出并放在冰上自然融化待用；
2.使用1×assay buffer(测试缓冲溶液)来配置实验所用中的“溶液A”(蛋白溶液)，“溶液B”(多肽溶液)，和“溶液C”(检测试剂溶液)，并使得各个组分在实验反应体系中，形成3×的溶液，溶液A、B、C的量须足够该轮实验所需量。
3)实验操作步骤：
实验板即实验前使用Echo非接触式纳升级声波移液***准备好的含有化合物梯度浓度和相应的DMSO溶液的板子：
a)取出实验板，并向实验板的第2～23列，加入5μL/孔的“溶液A”(蛋白溶液)，再向实验板的第1和24列加入5ul/孔的1×assay buffer，1和24列作为实验***中的Min control；
b)离心1000转，30秒；
c)将板子置于23℃孵育20分钟；
d)孵育20分钟之后，向实验板的1～24列加入5μL/孔的“溶液B”(多肽溶液)；
e)离心1000转，30秒；
f)将板子置于23℃孵育20分钟；
g)孵育20分钟之后，向实验板的1～24列加入5μL/孔的“溶液C”(检测试剂溶液)；
h)离心1000转，30秒；
i)将板子置于23℃孵育40分钟；
j)将实验板置于EnVision上读板。
4)数据分析：
a)用每块实验板的相应的Max control(最大控制)和Min control(最小控制)来换算出该实验板的Z’值，并确保每块板的Z’值>0.5；
b)对照化合物的信号通过XLFIT5计算出IC 50值，并确保其维持在历史数据平均值的3倍以内，结果见表5。
表1 BRD4检测IC 50测试结果
Figure PCTCN2021079672-appb-000003
5)结论：
化合物A对BRD4-BD1和BRD4-BD2均有显著抑制作用。
二、对小鼠移植瘤模型的体内药效试验
实施例2：化合物A在人乳腺癌MDA-MB-231_luc细胞皮下异种移植肿瘤模型的体内药效研究
1.实验动物与分组给药
1.1实验动物
种属：小鼠
品系：BALB/c裸小鼠
周龄及体重：6-8周龄，体重18-22克
性别：雌性
供应商：上海西普尔-必凯实验动物有限公司
1.2受试物配制方法
表2 受试物配制方法
Figure PCTCN2021079672-appb-000004
注：BID：每天两次。
1.3分组与给药方案
表3 体内药效实验动物分组及给药方案
Figure PCTCN2021079672-appb-000005
注：PO：口服灌胃。
2.实验方法与步骤
2.1细胞培养
人乳腺癌MDA-MB-231_luc细胞体外单层培养，培养条件为RPMI-1640培养基(供应商：Gibco；货号:22400-089；生产批号：4868546)中加10％胎牛血清，100U/ml的盘尼西林和100μg/ml的链霉素37℃ 5％CO 2培养。一周两次用胰酶-EDTA进行常规消化处理传代。当细胞处于指数生长期时，收取细胞，计数，接种。
2.2肿瘤细胞接种
将0.2mL 10×10 6个MDA-MB-231_luc细胞皮下接种于每只裸小鼠的右后背(PBS:Matrigel＝1:1)。肿瘤平均体积达到100-150mm 3时开始分组给药。
2.3肿瘤测量和实验指标
实验指标是考察肿瘤生长是否被抑制、延缓或治愈。每周两次用游标卡尺测量肿瘤直径。肿瘤体积的计算公式为：V＝0.5a×b 2，a和b分别表示肿瘤的长径和短径。
化合物的抑瘤疗效用TGI(％)或相对肿瘤增殖率T/C(％)评价。
TGI(％)，反映肿瘤生长抑制率。TGI(％)的计算：TGI(％)＝【(1-(某处理组给药结束时平均瘤体积－该处理组开始给药时平均瘤体积))/(溶剂对照组治疗结束时平均瘤体积－溶剂对照组开始治疗时平均瘤体积)】×100％。
相对肿瘤增殖率T/C(％)计算公式如下：T/C％＝T RTV/C RTV×100％(T RTV：治疗组RTV；C RTV：阴性对照组RTV)。
根据肿瘤测量的结果计算出相对肿瘤体积(relative tumor volume，RTV)，计算公式为RTV＝V t/V 0，其中V 0是分组给药时(即d 0)测量所得平均肿瘤体积，V t为某一次测量时的平均肿瘤体积，T RTV与C RTV取同一天数据。
2.4统计分析
统计分析，包括每个组的每个时间点的肿瘤体积的平均值和标准误(SEM)。治疗组在试验结束时给药后第21天表现出最好的治疗效果，因此基于此数据进行统计学分析评估组间差异。两组间比较用T-test进行分析，三组或多组间比较用one-way ANOVA进行分析，如果F值有显著性差异，应用Games-Howell法进行检验。如果F值无显著性差异，应用Dunnet(2-sided)法进行分析。用SPSS 17.0进行所有数据分析。p<0.05认为有显著性差异。
3.实验结果
给药21天后，化合物A的抑瘤率TGI＝54.85％，T/C＝52.99％，与溶媒对照组比较，p＝0.200；动物体重无明显变化，具有良好的耐受性。
实施例3：化合物A在人***癌PC-3细胞皮下异种移植肿瘤模型的体内药效研究
1.实验设计
受试物配制方法同表2，动物分组及给药方案同表3。
2.实验动物
种属：小鼠
品系：BALB/c裸小鼠
周龄及体重：6－8周龄，体重18-22克
性别：雄性
供应商：上海西普尔-必凯实验动物有限公司
3.实验方法与步骤
3.1细胞培养
人***癌PC-3细胞体外单层培养，培养条件为F-12K培养基(供应商：Gibco；货号: 21127-022；生产批号：1868870)中加10％胎牛血清，100U/mL的盘尼西林和100μg/mL的链霉素37℃ 5％CO 2培养。一周两次用胰酶-EDTA进行常规消化处理传代。当细胞处于指数生长期时，收取细胞，计数，接种。
3.2肿瘤细胞接种
将0.1mL 10×10 6个PC-3细胞皮下接种于每只裸小鼠的右后背。肿瘤平均体积达到100-150mm 3时开始分组给药。
3.3肿瘤测量、实验指标和统计分析同MDA-MB-231模型试验。
4.实验结果
给药21天后，受试化合物A与溶媒对照组相比，具有显著的抑瘤作用(T/C＝44.63％，TGI＝58.4％，p＝0.033)；动物具有良好的耐受性。
实施例4：化合物A在MC38小鼠结肠癌细胞动物移植瘤模型的体内抗肿瘤药效研究
1.实验设计
受试物配制方法同表2，动物分组及给药方案见下表4。
表4 体内药效实验动物分组及给药方案
Figure PCTCN2021079672-appb-000006
2.实验动物
种属：小鼠
品系：C57BL6小鼠
周龄及体重：6～7周龄，体重16-20克
性别：雌性
供应商：上海斯莱克实验动物有限责任公司
3.实验方法与步骤
3.1细胞培养
小鼠结肠癌MC38细胞(和元生物技术股份有限公司)体外单层培养，培养条件为10％胎牛血清的DMEM培养基(Gibco，货号：12100)，37℃ 5％CO 2的培养箱中培养。用0.25％ 胰酶-EDTA进行常规消化处理传代。当细胞处于指数生长期，饱和度为80％～90％时，收取细胞，计数，接种。
3.2肿瘤细胞接种
将0.1mL 2×10 5个MC38细胞皮下接种于每只小鼠的右后背，待肿瘤平均体积达到约70mm 3时，根据肿瘤体积进行随机分组给药。
3.3肿瘤测量、实验指标和统计分析同MDA-MB-231模型试验。
4.实验结论
给药20天后，与溶媒对照组比较，化合物A的15mg/kg给药组相对肿瘤增殖率T/C＝33.68％，肿瘤生长抑制率TGI＝68.81％，p<0.0001；25mg/kg给药组相对肿瘤增殖率T/C＝27.59％，TGI＝75.21％，p<0.0001；50mg/kg给药组T/C＝10.04％，TGI＝93.46％，p<0.0001。各给药组动物具有显著的抑瘤作用以及良好的耐受性。
三、毒理学研究
实施例5 大鼠单次给药毒性试验
40只SD大鼠，雌、雄各半，分为2组，每组20只，10只/性别/组，组别为溶媒对照组和化合物A组。化合物A组单次给药剂量为100mg/kg，单次给药容积为10mL/kg，给药浓度为10mg/mL(最大可灌胃浓度)，24h内给药2次(间隔8小时)，总给药剂量为200mg/kg。溶媒对照组给予等体积溶媒。
试验期间，化合物A给药组动物未见严重中毒症状，摄食、血液指标、凝血指标、大体解剖观察均未见异常。200mg/kg的化合物A对SD大鼠消化***可能存在可逆的毒性作用，可致动物流涎、水样便及雌性体重增长略缓。化合物A给药组雄性动物血清UREA水平减低。因此，本试验条件下，SD大鼠单次经口灌胃给予化合物A的最大耐受剂量(MTD)为200mg/kg。
实施例6 犬单次给药毒性试验
8只Beagle犬，雌、雄各半，分性别按体重随机分4组，每组2只，雌、雄各半。组别为：溶媒对照组、化合物A低剂量组、化合物A中剂量组和化合物A高剂量组。化合物低、中、高剂量组单次给药剂量分别为6mg/kg、18mg/kg和48mg/kg，24h内给药2次(间隔8小时)，总给药剂量分别为12mg/kg、36mg/kg和96mg/kg。溶媒对照组给予等体积溶媒。
试验期间，化合物A各给药组动物未见严重中毒症状，仅在给药当天第二次给药后出现一过性水样便，高剂量组动物在给药后4～5天食量有下降的趋势。化合物A各剂量组 Beagle犬给药后血液指标WBC和#NEUT有升高趋势。因此，本试验条件下，化合物A单次给药对Beagle犬的无毒性反应剂量(NOAEL)为36mg/kg；MTD为96mg/kg。
实施例7 SD大鼠重复给药4周恢复4周伴随TK毒性试验研究
SD大鼠分为4组，每组46只/组(主试验30只/组，TK卫星组16只/组)，雌雄各半。组别为溶媒对照组、化合物A低剂量组、化合物A中剂量组、化合物A高剂量组。化合物A低、中、高剂量组SD大鼠每天2次(间隔8小时)经口灌胃给予2，6和20mg/kg的化合物A(总给药剂量分别为4、12、40mg/kg·day)连续4周，停药恢复4周。溶媒对照组给予等体积溶媒。
结果显示：给药期间，包括溶媒对照组在内的各组别动物均出现间歇性软便，化合物A中剂量组和高剂量组的SD大鼠间歇性流涎，高剂量组个别SD大鼠还可见肛周污秽、自发活动减退、毛色黄、尿液颜色异常及消瘦。高剂量组雄性动物摄食减少、体重增长缓慢，停药后可恢复。临床病理检查提示供试品中、高剂量时对血液***、凝血***具有可逆的毒性作用，具体表现为NEUT和RETIC升高、LYMPH和/或PLT降低，PT延长、TT缩短。组织病理学检查发现12～40mg/kg的化合物A对雌、雄SD大鼠消化***、免疫***、呼吸***、骨骼***具有可逆的毒性作用，表现为直肠粘膜杯状细胞减少，脾脏和骨髓淋巴细胞减少，胸腺重量/脏器系数下降，肺脏泡沫巨噬细胞聚集，股骨、膝关节骨小梁增加。40mg/kg的化合物A对雄性SD大鼠心脏具有可逆的毒性作用，表现为单核细胞浸润或心肌坏死。
TK结果显示D1给药后，化合物A中剂量组AUC0-24h存在明显性别差异，D1和D28给药后，其余各剂量组的***暴露量(AUC0-24h和Cmax)均无明显性别差异；D1和D28给药后，雌性动物化合物A的***暴露量(AUC0-24h和Cmax)呈剂量比例性增加；雄性动物化合物A的AUC0-24h增加比例高于剂量的增加，Cmax呈剂量比例性增加；每天给药2次，连续给药4周后，雌、雄SD大鼠血浆中化合物A暴露量均无蓄积。
因此，本试验条件下，MTD为40mg/kg/day。该剂量下雄性和雌性动物第28天的AUC0-24h分别为23115和18017h*nM。NOAEL为4mg/kg/day。
实施例8 比格犬重复给药28天恢复28天伴随TK毒性试验研究
比格犬随机分为4组，10只/组，雌雄各半。组别为溶媒对照组、化合物A低剂量组、化合物A中剂量组、化合物A高剂量组。化合物A低、中、高剂量组每天2次(间隔8小 时)经口灌胃给予1，3和6mg/kg的化合物A(每天总给药剂量分别为2mg/kg、6mg/kg和12mg/kg)连续4周，停药恢复4周。
结果显示：给药期间，12mg/kg化合物A剂量组有3/10例动物(1雌2雄)濒死，死亡原因与供试品消化***、血液***和免疫***等毒性有关。化合物A各剂量给药后对动物消化***存在毒性作用，表现为给药后动物出现呕吐、粪便异常、食欲下降、体重增长缓慢或下降，以中、高剂量组动物的毒性反应明显。高剂量化合物A还可致动物活动减退、消瘦、震颤、步态异常、眼或鼻分泌物异常。高剂量和/或中剂量化合物A影响Beagle犬血液学指标(RETIC、EOS、BASO降低)、凝血指标(APTT延长、FIB升高)和血生化指标(TP、ALB、CREA、UREA降低，ALT、AST、CHOL、TG、GLU升高)，但作用可逆。组织病理学检查发现2mg/kg的化合物A对Beagle犬免疫***(胸腺)具有可逆的毒性作用。6～12mg/kg的化合物A对Beagle犬消化***(胰腺、肝脏、消化道)、免疫***(胸腺/脾脏、肠道相关***)、血液***(胸骨骨髓)及皮肤具有可逆的毒性作用，对Beagle犬生殖***(睾丸、附睾)的毒性作用停药后未完全恢复，恢复期动物可见脾脏髓外造血。12mg/kg的化合物A对Beagle犬呼吸***(气管、肺脏)有可逆的毒性作用。
TK结果显示D1和D28给药后，各剂量组化合物A***暴露量(AUC0-24h和Cmax)均无明显性别差异；重复给药后各剂量组化合物A暴露量(AUC0-24h和Cmax)均无明显蓄积；D1和D28给药后，化合物A的AUC0-24h增加比例均高于剂量的增加，Cmax均呈剂量比例性增加。
因此，本试验条件下，致死剂量为12mg/kg/day，HNSTD(最高非严重毒性剂量)为6mg/kg/day。该剂量下雄性和雌性动物第28天的(AUC0-24h)分别为9443.7和9149.9h*nM。观察到有害作用的最低剂量(LOAEL)为2mg/kg/day。
四、临床研究
实施例9 Ⅰ期临床试验研究
1.方案设计
本试验为一项针对晚期实体瘤患者的开放、剂量递增及队列扩展的Ⅰ期临床研究，旨在评价化合物A在晚期实体瘤患者中的安全性、耐受性、药代动力学特征以及初步抗肿瘤活性。终点指标包括：
(1)安全性：AE、SAE、DLT发生情况和频率；确定化合物A的最大耐受剂量(MTD)(如有)、II期临床推荐剂量(RP2D)和给药方案。
(2)药代动力学指标：包括但不限于AUC 0-last、AUC 0-∞、C max、T max、t 1/2和CL/F等。
(3)疗效指标：总缓解率(ORR)、无进展生存期(PFS)、疾病控制率(DCR)、缓解持续时间(DOR)等。
本研究分两阶段进行，第一阶段(Stage I)为剂量递增(Dose escalation)和剂量扩增(Dose expansion)研究，第二阶段(Stage II)为队列扩展(Extension Cohort)研究。
第一阶段(Stage I)：
剂量递增研究：
参考前期毒理试验中HNSTD/MTD剂量和体内药效试验剂量，设计临床研究剂量。化合物A的起始给药剂量定为20mg/天，初步拟定5个剂量组：20、40、80、120、160mg。预设的最高递增剂量为160mg/天。本研究将遵循“i3+3”剂量递增方案。
每个剂量水平第一周期(31天)，受试者第1天单次给药后(第1天)，观察3天未出现DLT及严重不良事件，则在第4天进入连续给药阶段。(具体的给药频率将根据患者的耐受性、安全性及药物PK参数进行调整)。
完成第一周期(Cycle 1)给药后，评估受试者的安全性、耐受性、药代动力学特征及抗肿瘤活性。如受试者耐受性良好且本人同意，则将继续随后周期(Cycle 2-Cycle N)每28天的重复周期给药，直至出现疾病进展或不可耐受毒性，或其他原因终止研究。
剂量扩增研究：
可根据剂量递增研究获得的安全性、耐受性及有效性等数据，必要时进行目标剂量组剂量扩增研究和不同给药方案(如给药21天，停药7天)的探索研究。申办者和研究者将持续进行安全性评价，基于既往剂量水平的可用数据，确定剂量递增和剂量扩增期间的用药剂量水平和给药方案。
第二阶段(Stage II)：
在第一阶段确定MTD、初步预估的RP2D以及合适的给药方案后，将在不同适应症中进行队列扩展研究，以观察研究药物的安全性、耐受性、药代动力学特征和抗肿瘤活性。
扩展阶段可能选定不同瘤种的人群，每个扩展人群预计入组20～40例受试者。最终的队列扩展的样本量和扩展瘤种将综合考虑第一阶段和临床前研究数据，在与研究者充分讨论后确定。并且，申办者和研究者在基于患者最大获益/风险比的原则下可考虑提前结束某队列的入组。
以上两个阶段的Ⅰ期临床实验，共拟纳入恶性晚期实体瘤患者53-186例。
2.入选与排除标准
1)入选标准
有资格参加本次研究的受试者必须符合下列各条入选标准：
1.年龄18～75(含)周岁，性别不限。
2.入组受试者必须有经组织学或细胞学确诊的晚期或转移性肿瘤，且无标准治疗方案，或对标准治疗方案无效或不耐受者，或没有条件接受标准治疗，并符合以下相应阶段的瘤种要求：
(1)Stage I：不限实体瘤瘤种；
(2)Stage II：
①Arm 1：TNBC
②Arm 2：CRC
③Arm 3：其他可能的瘤种
3.至少有一个可测量的病灶，可测量的定义源于RECIST 1.1版标准。
4.ECOG体能状态评分：0～1分。
5.预计生存时间超过3个月。
6.主要器官功能在治疗前7天内，符合下列标准(在研究药物首次给药前14天内未接受过输血、EPO、G-CSF或其他医学支持治疗)：
Figure PCTCN2021079672-appb-000007
7.女性应为同意在研究期间和研究结束后6个月内必须采用避孕措施(如宫内节育器[IUD]，避孕药或避孕套)；在研究拟入组前的7天内血清妊娠试验阴性，且必须为非哺乳期受试者；男性应同意在研究期间和研究期结束后6个月内必须采用避孕措施的受试者。
8.受试者须在试验前对本研究知情同意，并自愿签署书面的知情同意书。
2)排除标准
以下任何一条均可将受试者剔除，不入选研究：
1.在首次使用研究药物前4周内接受过化疗、放疗、生物治疗、内分泌治疗、靶向治疗、免疫治疗等抗肿瘤治疗，除外以下几项：亚硝基脲类(如卡莫司汀、洛莫司汀等)或丝裂霉素C为首次使用研究药物前6周内；口服氟尿嘧啶类、小分子靶向药物为首次使用研究药物前2周或已知药物的5个半衰期内(以时间长的为准)；有抗肿瘤适应症的中药为首次使用研究药物前2周内。
2.在首次使用研究药物前4周内接受过其它未上市的临床研究药物治疗。
3.在首次使用研究药物前4周内接受过主要脏器外科手术(不包括穿刺活检)或出现过显著外伤。
4.在首次使用研究药物前14天内接受过全身使用的糖皮质激素(强的松＞10mg/天或等价剂量的同类药物)或其他免疫抑制剂治疗；除外以下情况：使用局部、眼部、关节腔内、鼻内和吸入型糖皮质激素治疗；短期使用糖皮质激素进行预防治疗(例如预防造影剂过敏)。
5.在首次使用研究药物前14天内使用过CYP3A4的强效抑制剂或强效诱导剂者。
6.既往曾接受过BET抑制剂治疗。
7.既往抗肿瘤治疗的不良反应尚未恢复到CTCAE 5.0等级评价≤1级(脱发等研究者判断无安全风险的毒性除外)。
8.具有临床症状的中枢神经***转移或脑膜转移，或有其他证据表明患者中枢神经***转移或脑膜转移灶尚未控制，经研究者判断不适合入组。
9.不能控制的活动性感染(急性或慢性真菌，细菌，病毒或其他感染)。
10.有自身免疫性疾病史、免疫缺陷病史，包括HIV检测阳性，或患有其他获得性、先天性免疫缺陷疾病，或有器官移植史。
11.活动性乙型肝炎(乙型肝炎病毒滴度＞1000拷贝/ml或200IU/ml)，允许除干扰素以外的预防性抗病毒治疗；丙型肝炎抗体阳性。
12.有严重的心血管疾病史，包括但不限于：
(1)有严重的心脏节律或传导异常，如需要临床干预的室性心律失常、Ⅱ～Ⅲ度房室传导阻滞等，或需服用抗心律失常药物的其他心律失常(不包括抗凝药物，服用抗凝药物相关凝血功能必须满足入选标准)；
(2)有心肌梗塞、心绞痛、血管成形术、冠状动脉架桥外科病史；
(3)基线期心电图QT/QTc间期延长者(QTcF>480ms)；
(4)基线超声心动图(ECHO)或多门控采集(MUGA)技术显示左室射血分数(LVEF)≤50％；
(5)心力衰竭，纽约心脏病学会(NYHA)分级为II级及以上；
(6)控制不良的高血压(尽管使用了最优治疗，但收缩压≥150mmHg和/或舒张压≥95mmHg)；
(7)既往或当前患有心肌病。
13.无法口服吞咽药物，或存在经研究者判断严重影响胃肠道吸收的状况。
14.有其它严重的***性疾病史，经研究者判断不适合参加临床试验的患者。
15.已知有酒精或药物依赖。
16.既往有明确的神经或精神障碍史，包括癫痫或痴呆。
17.妊娠期或哺乳期女性。
18.研究者认为受试者存在其他原因而不适合参加本临床研究。
3.研究结果
目前临床试验进行中，受试者例数较少，治疗周期短，尚不足以对最终结果以及后期方案做出准确预估。综合考虑毒理试验耐受剂量、非临床药效学剂量及同类化合物I期临床剂量，预计化合物A最大耐受剂量(MTD)为120mg/d～160mg/d，预期疗效剂量为80mg/d。在试验条件下，化合物A给药80mg/d、120mg/d、160mg/d，将对晚期实体瘤，尤其是胃肠肿瘤、三阴乳腺癌、结直肠癌、***癌患者产生有效的治疗作用。研究中，如果剂量递增至160mg/d时，该剂量组安全性和耐受性仍良好，则可由研究者和申办者共同讨论决定是否进行更高剂量的探索。更高剂量的确定将基于已有剂量水平的可用数据，目前尚无法准确预估。
可以预期，化合物A将能安全有效的治疗晚期实体瘤，尤其是胃肠肿瘤、三阴乳腺癌、结直肠癌、***癌。
实施例10 典型病例
某患者，男性，62岁，2015年诊断直肠癌IIIB期，行腹腔镜下直肠癌前切根治术，术后辅助奥沙利铂+5-Fu、术后辅助盆腔放疗；后经历过奥沙利铂+卡培他滨、FOLFIRI(伊立替康+5-Fu+亚叶酸钙)、希明替康+沙利度胺、卡培他滨单药、重组抗EGFR人鼠嵌合单抗隆抗体注射液、瑞戈非尼治疗；2017年针对左肺病变两次放疗。均发生病情进展，治疗失败。患者入组时诊断：直肠癌IVA期。
该患者于2020-5-13第一次给予化合物A口服，剂量20mg。观察两天无严重异常，于2020-5-15继续给药，剂量为20mg/天，末次服药2020-7-10，共服药2个周期(每周期28 天)。第二周期末做增强CT检查，综合评估为SD(疾病稳定)。用药期间发生过1级低白蛋白血症、1级贫血、1级白细胞计数降低。低蛋白血症与研究药物可能无关，后两者与研究药物可能有关。
说明书英文缩略词的全称和中文释义如下：
缩写和术语 英文全称 中文释义
AE Adverse event 不良事件
AUC Area under drug time curve 药时曲线下面积
AL Acute leukemia 急性白血病
ALT Alanine aminotransferase 丙氨酸氨基转移酶
ANC Absolute neutrophil count 中性粒细胞计数
APTT Activeated partial thromboplastin time 部分凝血活酶时间
AR Androgen receptor 雄激素受体
AST Aspartate aminotransferase 天门冬氨酸氨基转移酶
BET Bromodomain and extra-terminal domain 溴结构域及超末端结构
BID Bis in die 每日两次
BRCA Breast cancer susceptibility gene 乳腺癌易感基因
BRD4 Bromodomain-containing protein 4 溴结构域蛋白4
CHL Chinese hamster lung cells 中国仓鼠肺细胞
CHO Chinese Hamster Ovary 中国仓鼠***
CL Plasma clearance rate 血浆清除率
Cmax Peak concentration 峰浓度
CR Complete remission 完全缓解
CRC Colorectal cancer 结直肠癌
DFS Disease-free survival 无病生存期
DLBCL Diffuse large B-cell lymphoma 弥漫性大B细胞淋巴瘤
DLT Dose limited toxicity 剂量限制毒性
EC50 50％effective concentration 半数有效浓度
ECHO Echocardiography 超声心动图
ER Estrogen receptor ***受体
GPCR G protein coupled receptor G蛋白偶联受体
HER2 Human epidermal growth factor receptor 2 人表皮生长因子受体
HNSTD Maximum non serious toxic dose 最高非严重毒性剂量
ICF Informed consent form 知情同意书
INR International Normalized Ratio 国际标准化比值
IUD Intrauterine device 宫内节育器
LOAEL The lowest dose at which harmful effects are observed 观察到有害作用的最低剂量
MTD Maximum torlerate dose 最大耐受剂量
MUGA Multiple uptake gated acquisition scan 多门电路探测
NOAEL No harmful effect dose level 未见有害作用剂量水平
NYHA New York Heart Association 美国纽约心脏病协会
OHM Other hematological malignancies 其他血液***恶性肿瘤
OS Overall survival 总生存期
PD-L1 Programmed cell death-Ligand 1 程序性死亡受体-配体1
PR Partial remission 部分缓解
PD progression disease 疾病进展
PR Progesterone receptor 孕激素受体
PT Prothrombin time 凝血酶原时间
RECIST Response evaluation criteria in solid tumours 实体瘤疗效评价标准
RP2D Recommended phase 2 dose Ⅱ期临床试验推荐剂量
SAE Serious adverse event 严重不良事件
SD Stable disease 疾病稳定
TGI Tumor growth inhibition 肿瘤生长抑制
T1/2 half life 半衰期
Tmax Peak time 达峰时间
TNBC Triple-Negative Breast Cancer 三阴乳腺癌
ULN Upper limit of normal 正常值上限
Vdss Apparent volume of steady state distribution 稳态表观分布容积

Claims (14)

  1. 化合物A或其药学上可接受的盐在制备***的药物中的应用，所述化合物A的结构如下式(Ⅰ)所示：
    Figure PCTCN2021079672-appb-100001
    所述肿瘤优选晚期实体瘤或难治性实体瘤，更优选所述晚期实体瘤或难治性实体瘤中的实体瘤为胃肠肿瘤、结直肠癌、乳腺癌或***癌，进一步优选所述晚期实体瘤或难治性实体瘤中的实体瘤为肿瘤相关巨噬细胞高度浸润的结直肠癌、三阴乳腺癌、雄激素非依赖型***癌；优选患有所述肿瘤的对象为人。
  2. 如权利要求1所述的应用，其特征在于：所述药物制成临床接受的制剂，例如口服制剂、注射制剂、局部给药制剂、外用制剂。
  3. 如权利要求1或2所述的应用，其特征在于：所述药物为单剂量剂型或多剂量剂型；所述剂型中含有治疗有效量的化合物A或其药学上可接受的盐(以化合物A的重量计)，约0.001mg-约1000mg，优选约1mg-约500mg，或约5mg-约400mg，或约10mg-约300mg，或约10mg-约250mg，或约10mg-约200mg，或约20mg-约200mg，更优选约10mg-约160mg，或约20mg-约160mg，或者约80mg-约160mg；例如约10mg、约20mg、约40mg、约60mg、约80mg、约100mg、约120mg、约140mg、约160mg，更优选约10mg、约20mg、约40mg、约80mg、约120mg或约160mg，进一步更优选约20mg、约40mg、约80mg、约120mg或约160mg，或者进一步更优选约10mg或约40mg。
  4. 如权利要求1-3中任一项所述的应用，其特征在于：所述化合物A或其药学上可接受的盐可与其他靶向药物或化疗药物中的一种或多种联合使用。
  5. 一种***的方法，所述方法包括给予受试者或患者治疗有效量的化合物A或其药学上可接受的盐，所述化合物A的结构如下式(Ⅰ)所示：
    Figure PCTCN2021079672-appb-100002
    所述肿瘤优选晚期实体瘤或难治性实体瘤，进一步优选所述晚期实体瘤或难治性实体瘤中的实体瘤为胃肠肿瘤、乳腺癌、结直肠癌或***癌，进一步优选所述晚期实体瘤或难治性实体瘤中的实体瘤为肿瘤相关巨噬细胞高度浸润的结直肠癌、三阴乳腺癌、雄激素非依赖型***癌；优选所述受试者或患者为人。
  6. 如权利要求5所述的方法，其特征在于，所述给予是口服给予、注射给予、局部给予或体外给予；或者化合物A或其药学上可接受的盐制成临床接受的制剂后给予，所述制剂包括口服制剂、注射制剂、局部给药制剂、外用制剂。
  7. 如权利要求5-6中任一项所述的方法，其特征在于，化合物A或其药学上可接受的盐(以化合物A计)每天给药剂量为约0.001mg-约1000mg，优选约1mg-约500mg，或约5mg-约400mg，或约10mg-约300mg，或约10mg-约250mg，或约10mg-约200mg，或约20mg-约200mg，更优选约10mg-约160mg，或约20mg-约160mg，或者约80mg-约160mg；例如约10mg、约20mg、约40mg、约60mg、约80mg、约100mg、约120mg、约140mg、约160mg，更优选约10mg、约20mg、约40mg、约80mg、约120mg或约160mg，进一步更优选约20mg、约40mg、约80mg、约120mg或约160mg，以单剂量或分剂量施用。
  8. 如权利要求5-7中任一项所述的方法，其特征在于，化合物A或其药学上可接受的盐的给药方案为每个周期28天，每天给药一次，给药21天，停药7天；优选每次给予化合物A或其药学上可接受的盐(以化合物A计)约20mg、约40mg、约80mg、约120mg、约160mg。
  9. 如权利要求5-8中任一项所述的方法，其特征在于：所述化合物A或其药学上可接受的盐与其他靶向药物或化疗药物中的一种或多种联合使用。
  10. 化合物A或其药学上可接受的盐，用于***，所述肿瘤优选晚期实体瘤或难治性实体瘤，更优选所述晚期实体瘤或难治性实体瘤中的实体瘤为胃肠肿瘤、结直肠癌、乳腺癌或***癌，进一步优选所述晚期实体瘤或难治性实体瘤中的实体瘤为肿瘤相关巨 噬细胞高度浸润的结直肠癌、三阴乳腺癌、雄激素非依赖型***癌；优选患有所述肿瘤的对象为人，
    其中所述化合物A的结构如下式(Ⅰ)所示：
    Figure PCTCN2021079672-appb-100003
  11. 如权利要求10所述的化合物A或其药学上可接受的盐，其特征在于，所述化合物A或其药学上可接受的盐制成临床接受的制剂后给予，所述制剂包括口服制剂、注射制剂、局部给药制剂、外用制剂。
  12. 如权利要求10-11中任一项所述的化合物A或其药学上可接受的盐，其特征在于，化合物A或其药学上可接受的盐(以化合物A计)每天给药剂量为约0.001mg-约1000mg，优选约1mg-约500mg，或约5mg-约400mg，或约10mg-约300mg，或约10mg-约250mg，或约10mg-约200mg，或约20mg-约200mg，更优选约10mg-约160mg，或约20mg-约160mg，或者约80mg-约160mg；例如约10mg、约20mg、约40mg、约60mg、约80mg、约100mg、约120mg、约140mg、约160mg，更优选约10mg、约20mg、约40mg、约80mg、约120mg或约160mg，进一步更优选约20mg、约40mg、约80mg、约120mg或约160mg，以单剂量或分剂量施用。
  13. 如权利要求11-12中任一项所述的化合物A或其药学上可接受的盐，其特征在于，化合物A或其药学上可接受的盐的给药方案为每个周期28天，每天给药一次，给药21天，停药7天；优选每次给予化合物A或其药学上可接受的盐(以化合物A计)约20mg、约40mg、约80mg、约120mg、约160mg。
  14. 如权利要求11-13中任一项所述的化合物A或其药学上可接受的盐，其特征在于：所述化合物A或其药学上可接受的盐与其他靶向药物或化疗药物中的一种或多种联合使用。
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